ORVl LLE WYSS University of Texas, Austin, Tex.
Microbial Adaptation The limits of microbial adaptation are wide. Much of it has occurred in the past when organisms developed that approximately fit any existing microenvironment. Because of their small size and their independent existence microorganisms are unable to give significant protection to their cytoplasm or germ plasm, and these must respond to environmental challenge if the organism is to survive.
MICROBIAL
adaptations can be discussed from a variety of approaches, and general reviews on the subject are available (2, 3, 4 ) . While specialists in the various phases of this field usually approach the problem by setting u p definitions of the term adaptation in its various phases, the familiar concept, which includes the adjustments microorganisms make or have made in some past time when confronted with a challenge to their existence, will be assumed here. Such adjustments, that have made or can make a certain segment of the totality of microbial life better able to survive and reproduce in a given environment, have been studied a t all levels. They range from those adjustments made by certain great groups such as the molds conferring on them greater fitness than processed by other groups in a low moisture environment to those made within a single bacterium which enables it to ferment a particular type of sugar. Adaptation on the species level is a problem in ecology and is treated later in this symposium by Weindling, p. 1407. I n practical problems it is often of paramount importance. When one considers the problem of microbial attack on a product, the control of one species often releases that environmental niche for occupancy by another group which is able to resist the applied inhibitory agent but had not been able to meet the competitive challenge from the normal inhabitants. T h e parasites are a special case where for the most part the challenge is from the various devices of the host which confer resistance rather than from competition with other organisms. Here adaptations occur which confer virulence on the successful competitor. But even in this field we find important instances of better fitness of one group than another
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as when certain antibiotics, which are administered to relieve a n infection, also destroy normal nonpathogenic bacteria in the ear canal or the bowel and release these sites to the occupancy of pathogenic fungi. Outstanding illustrations of adaptation on this level are encountered in the decomposition of organic residues. A species attacking the soluble sugars in a new compost pile grows so vigorously that before the sugars are gone the temperature has increased to a level where this population yields not to a thermophilic variant of the same species but to another species which now fits the environment for a short time until it too is displaced in its turn. The large number of microbial species extant makes it unlikely that a n environment exists in the compost to which some species is not relatively well adapted. And since the species of microorganisms involved in such processes are ubiquitous, we need not be concerned lest the proper organism fail to be present when a suitable environment arises. The difficulty in showing benefit from compost inoculation attests to this and the explanation apparently lies in the rapid multiplication of the microbes and in the fact that any clod of soil will contain millions of microenvironments-some aerobic, some anerobic, some high in nutrients and some low-and each microenvironment has its characteristic microflora. I n addition microbes can with few exceptions lie dormant for long periods when their particular microenvironment disappears. This characteristic of being ever present where suitable environments may occur and then yielding the environment to the species which has been selected by past evolutionary processes as best fitted appears to account for a large portion
INDUSTRIAL AND ENGINEERING CHEMISTRY
of microbial adaptation. It pla)s a major role in determining the result obtained in tests involving microbial deteriorations, in composting, in studies on soil samples to determine the capacity for nitration, or, as the oil exploration, studies which determine the ability to oxidize hydrocarbons. I t also poses one of our most difficult problems-that of translating laboratory results to practical problems where we are unable to know accurately the environment in the micro sphere occupied by the natural microbial population in which we are interested. We can be fairly certain that any set of characteristics shown by a species are adaptive in that they fit that organism better than any other to a n environment to which it has access now or has had access in its past history. There a r r characteristics of living organisms which have been termed nonadaptive, and it is often pointed out that such characters seem to maintain themselves and even increase in certain populations. Many biologists believe this to be a n expression of our ignorance of the true nature of the microenvironment and the selective force a t work there. An analogy in man concerns a trait of sickle cell anemia which appears in about 7% of the negro population and appears to be spreading in a belt around the world on both sides of the equator. With only this information it is impossible to explain how such a characteristic could be a useful adaptation until one obtains the further information that malaria parasites find difficulty in invading the sickle-shaped red cells. I t is then quite evident that in the “microenvironment” of the malaria belt, a seemingly destructive character gives a net survival advantage to its possessor. I n the same way as between groups
WATER P U R I F I C A T I O N or species, adaptation occurs as a result of variation between cultures within a species and individuals within a culture. This can be demonstrated even in cultures started from a single cell-a procedure which appears less important than formerly, since we recognize that a clump of cells was a single cell a few generations previously and that with the lavish cell multiplication and catastrophic destruction that takes place in microenvironments it is unlikely that cells that are far removed from a common ancestor would be often in contact with each other. In the isolation of a species from nature we choose a medium and conditions of growth which appear optimum for the species. Yet the medium, no matter how carefully planned, usually selects specific individuals from the natural environment as fittest to survive in the artificial environment. Often these grow better after a period of training involving repeated transfers on the culture medium which means the selection of adaptations of the original adaptations resulting in individuals of a “superior” growth type. Since this superior growth is observed only if the organisms that have it pass it on to their descendants (in microbiology individuals are not studied but only their massed progeny as colonies or cultures), often the resultant culture will be the progeny of one or a few unusual individuals in the original inoculum. The artificial system occurs in the test tube, where there is no competition from exogeneous species and strains, and can be magnified extensively there, since we can face the clone with challenges that under natural conditions would have been answered by yielding to another species. While many test tube experiments are caricatures of what occurs in nature they lead us to a better understanding of how the culture which occupies an environment enters it as only a n approximate fit and then, by further modifications within itself, adds to its past evolutionary adaptedness by current evolutionary adaptation which selects those individuals from a developing clone whose inherited characteristics fit it for most rapid occupation. I t must be appreciated a t this point that even in the abscnce of competing species the laboratory selective process fails to measure the “long time survival ability” of the selected strains; we have observed selected strains that are “fittest” in that they give the greatest turbidity in 24 hours in antibiotic broth, but they require transfer every 2 days or they die. At the present time it is generally accepted that the individuals that father the “fitter strains” arise as a result of a sudden change in the genetic make-up which is passFd on a t cell division.
Such changes in bacteria are called mutations and presumably occur in all of the more than 10,000 genes present in microorganisms a t a n average rate of about once in every 10,000,000 times for each cell division for each gene. The mutation rate of each gene is itself a n adaptation that has been determined by evolutionary challenge since, if it occurred too seldom, the organism would not be able to meet a changing environment and, if too often, too many cells would be lost in wasteful modifications. The mutation rate is itself modified by such environmental effects as radiations, concentration cf certain inorganic salts, and of metabolic products like peroxides. Even a t an average rate of 1 in 10,000,000 new mutants occurring a t every generation it would seem that after a relatively few generations all the organisms would be mutant in one or more characters. Yet actual experience shows that cultures transferred under constant conditions remain relatively stable over long periods, I t has been suggested that an equilibrium between mutants and nonmutants is attained in the wild type population governed on the negative side by the relatively slower growth of the mutants and on the positive side by the appearance of new mutations in each generation. I t has been pointed out (7) that a periodic selection occurs in bacterial populations when new mutants appear which are superior in growth to the wild type. They overgrow and eliminate the parental type together with the other accumulated mutants until they in turn are eliminated by the same process. Periodic selection occurs often enough to prevent the attainment of a true mutational equilibrium and therefore paradoxically results in relative stability in cultures. To complicate our problems man’g industrial and scientific activities arc changing the environment as never before. Organisms are confronted with many new chemical substances in conwntrations and under conditions not previously known. Some examples show that, a t least in regards to antibiotic resistance, wild type organisms are reasonably well established. Chloromycetin-resistant cultures occurred a t a high rate in hospitals where that drug was widely used several years ago. When the use of the drug declined temporarily the resistant strains declined being once more replaced by wild type. In this laboratory we failed in attempts to select penicillin-resistant pathogens that would outgrow wild type, but such negative experiments are not conclusive and we know of no reason why they should not be found if the search is sufficiently diligent. For unusual org-
anisms to arise it is necessary that large numbers of wild tvpe be confronted with the challenge and that selective processes be permitted to prevail. Under such conditions many cell divisions will occur thus increasing the likelihood that a mutant organism will arise that will meet the challenge-as when organisms are subjected to a mild concentration of a n inhibitor in a medium permitting good growth or supplied with a new source of organic matter toqether with a limiting amount of organic substance on which the organism is able to make some growth. By such methods it is relatively easy to isolate a drug-resistant strain or a mannitol fermenting strain of Micrococcus pyogenes. With some inhibitors a single transfer is required to produce a new culture with the new characteristic; presumably following a single mutation within an organism there developed the new stock. With penicillin apparently several transfers into increasingly high concentrations are required to 1 ield the fully resistant strains. indicating that a series of mutations are required to yield the mutant. Since each mutation occurs a t a rate of the order 1 X 10-7 per cell division it is evident that three simultaneous mutations would occur in a single organism once out of lo2’ cell divisions, and one is unlikely to observe that many in a lifetime. This suggests that penicillin operates on several different sites in the organism and that a mutation a t the most sensitive site results in a mutant which can grow in a penicillin medium unless the concentration is increased to a level where another cell process is inhibited. A mutation in the gene controlling this process produces further resistance and this can be repeated in the test tube until the oiganism is resistant to over a thousand units. Such organisms with compound mutations are markedly different from the original wild type in other respects than in drug resistance and have little or no ability to survive in nature. Mutants with great resistance to antibacterial substances are obtained when the substances are specific in their mechanism of action-that is, when they interfere with one or only a few cell processes. Ll’ith substances that have a general antiprotoplasmic action such as acids, salt, surface active agents, and phenols, it is very difficult to secure mutants that will resist much increase in the destructive concentration. The active cell components are so similar in their reactivity to these substances that a mutation to resistance in the most sensitive site will permit the organism to withstand only a tiny percentage increase in concentration of the nonspecific poison before another cell process fails. VOL. 48, NO. 9
SEPTEMBER 1956
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When mutations giving any large degree of resistance to such general agents are
found they may be expected to involve a mechanism of keeping the lethal agent away from the active cell substance. Examples are detergent-resistant organisms that produce a lipoid capsule which protects the fats and proteins of the cell membrane and protoplast from contact with the agent and the radiation-resistant organism which produces higher amounts of the enzyme, catalase, to destroy the lethal peroxide produced by the radiation. Sometimes increased resistance is due to a n artifact in the test procedure. In the phenol coefficient test the selection o f a mutant that gives a higher population in 24 hours in the medium used to grow the stock will result a n apparent increase in resistance because one then is measuring the concentration of chemical required to kill a greater number of organisms. Although coliform organisms have been subjected to killing by chlorine on a large scale for over 40 years there is no evidence that there has been any adaptation to great tolerance. Although individual cells must vary somewhat in their resistance to chlorine this is not sufficient to produce any enrichment of organisms which have acquired heritable resistance to chlorine The straight line which results rvhen the log of survivors is plotted against time suggests rhat the last survivors are chance escapees of the lethal process rather than more resistant organisms. Only when the killing curve changes in slope as it approaches the .Y axis can it be concluded that the population may have been composed of organisms of sufficiently mixed resistance that it would be profitable to select the last survivors to determine if the resistance is of the inherited t\pe rather than a result of older cells. spores, etc. If the resistance is inherited. the killing curve of a population grown from the resistant mutant would have a modified slope. It is possible to secure organisms resistant to the bactericidal action of some of these agents by selecting mutants to the bacteriostatic action of the same chemical. However phenol-resistant mutants are resistant to the germicidal action of phenol only when grown in a broth containing a subinhibitory amount of phenol. Since the wild type would not grow in broth with equivalent phenol concentration it appears that there is a phenol-resistant mutation on which is superimposed some further adaptation of the organism. This suggests that for control of microorganisms in a natural environment a chemical that does not persist may have some advantage not heretofore recognized. I n addition to past and current evolutionary adaptation in which the course and fine adjustments bem-een the organism and the environment are made, a superfine adjustment also occurs.
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This takes place within the individual, for even the microbe as it emerges as an entity into this world cannot be exactly and rigidly adjusted to an environment since no tlvo organisms are ever confronted with exactly the same set of conditions. The major lines are drawn, but the details are left to be filled in; the organism makes “on-the-spot” adjustments within the fixed limits set by its heredity. This has been called physiological adaptation to distinguish it from evolutionary adaptation, and it is characterized by a modification of all rhe cells in a culture when confronted with a challenge and the return to the normal condition when the challenge is removed. Some of these modifications are direct as when a culture of any of a number of soil bacteria is exposed to penicillin. Each cell in the population begins to produce the enzyme penicillinase that destroys the penicillin. There is no selection; all cells are genetically endowed with the ability to produce the enzyme but do so only when the substrate is present. This is termed induced or adaptive enzyme formation. The production of an enzyme under a condition where it is useful and the ability to mulriply without the encumbrance of the unnecessary enzyme when it is of no value appears to be the basis for much of the on-the-spot adaptation available to the organism. Historically one of the best known examples is the enzyme in some coliforms that breaks up formic acid to hydrogen and carbon dioxide. This enzyme appears only afrer the cultures have been incubated for ‘ 2 hour in formic acid. The organisms in the culture need make no groJvth or cell division during the induction, indicating that this is not due to the selection of mutants. The environment changes thc organism though only within the limits set by its genetic pattern. In recent years there was developed a voluminous literature on induced enzymes and the effects are now well understood, but these are simple situations and man>- examples of environmental modifications that have adaptive value are much less straight-forward. The complexity can be appreciated when one considers thar the microbial cell carries on many series of interlocking reactions where the product of one enzyme reaction may be the substrate for several other enz>-mes: and any slight modificarion of even one reaction will produce modifications in diverse reactions many steps removed from the initial site, To use a n imaginary example, a cell is exposed to a n environment which slows down slightly the conversion of compound X to compound Y. Since compound Y is converted to 2 this means rhat not only Tvill there be less 2 available for the cell but also that X will pile up and perhaps reach a sufficient
INDUSTRIAL AND ENGINEERING CHEMISTRY
concentration that it can react with compound ‘4 to produce compound B, thus bleeding off compound -4 from its normal purpose in cell processes. The accumulation of compound B induces the formation of a new enzyme and leaves less enzyme building material to make the other enzymes in the cell. So the modifications pyramid when an individual cell is presented with even a minor environmental challenge. Many examples of such modifications can be presented but only a few illustrations are given of the problems these present to the experimentalist as well as the practical biologist. If cells of E. coli are soaked in salt solution for several hours this will have no effect on their resistance to killing by ultraviolet light. But if they are supplied with any one of several metabolites during this time all the cells in the population become more resistant to irradiation even though no growth has occurred. .I\ diligent search for the differences berween such cells and the normal population shows that lethal doses of ulrraviolet cause no leaching of nuclear substances from the salt-soaked cells whereas other cells lose nucleic acids rapidly following irradiation, It would appear that soaking the cell in salt in the presence of exogenous organic marter resulted in a cell whose membranes retained necessary cell constituents when these were disturbed by irradiation, and such cells did not die unless the radiation dose \vas increased so as to desrroy the next most sensitive site. I n microbial processes in industry it is \vel1 recognized that the production of a product may be affected by minor changes in environment, and the food concentration that is optimum for a given aeration level or p H must be modified if one or the orher of these is changed even slightly. I n soil processes it is reported that azotobacter, which require high oxygen tension for maximum nitrogen fixation when SUPplied with a 1% sugar solution, actually fixes nitrogen better under semianerobic conditions when the carbohydrate concentration is reduced to near that found in the soil.
Literature Cited ( 1 ) Atwood, K. C., Schneider, L. K., Ryan, F. J., Proc. LVatl. Acad. C. S. 37, 146-i3 (1951). ( 2 ) Davies, R., Gale, E. F., “Adaptation 3%.
hficroorganisms,‘’ Cambridge University Press. Cambridge, Eng., 1953. (3) Spiegelman, S., Landman, 0 . E., .4nn. Re?. .Microbid. 8 , 181--236, 1954. (4) Wagner, R. P., Mitchell, H. K.? “Genetics and ?rZetabolism,” John Wiley, S e w York, 1955. in
RECEIVED for review January 12, 1956 ACCEPTEDApril 12> 1956
